Measure substrate disappearance with amylase
Amylase progress can be followed by sampling for starch at fixed times and recording when it is no longer detected.
AS Level · Cambridge International Biology 9700, 2028–2030. These lessons teach enzyme concepts and experimental reasoning. Written practice is self-checked; it does not demonstrate supervised practical competence or replace teacher-reviewed assessment.
Before you begin
- Recognise that proteins have specific three-dimensional shapes and that reaction rates can be compared using controlled measurements.
Work on paper. Try each question before revealing help, and explain why your method works.
Quick readiness check
Which reagent can indicate whether starch remains in a sample?
What you will learn
- Describe how starch disappearance can be followed in an amylase reaction
- Use timed samples to compare reaction progress fairly
Use iodine as a starch indicator
Amylase catalyses starch hydrolysis. To follow substrate disappearance, remove equal small samples at fixed time intervals and add each to iodine solution using the same method. Blue-black indicates detectable starch; orange-brown indicates that starch is not detected by this test.
Use a consistent sampling interval, sample volume and iodine amount. The first sample with no blue-black result gives an interval estimate for when starch fell below the test’s detection, not an exact molecular endpoint.
Control the reaction conditions
Use the same starch and amylase concentrations, temperature, pH and volumes when comparing runs. A buffer can help maintain pH. Start timing consistently when enzyme and substrate are mixed.
The time to disappearance can compare progress under the specified conditions, but it is not automatically a full rate curve. More frequent sampling near the endpoint improves the time resolution. Repeat trials where possible and compare like with like.
Pause and explain
What does an orange-brown iodine result indicate in this assay?
Worked example
Samples taken every 20 s give a blue-black iodine result at 40 s and an orange-brown result at 60 s. What can be concluded?
Show the worked solution
- Starch is still detected at 40 s.
- It is not detected at 60 s, so disappearance occurred within the interval.
Answer Starch was detectable at 40 s but not detected at 60 s. Under this test, the starch fell below the detection level sometime after 40 s and by 60 s; the exact time is not known.
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Write your answer and reasoning first. Use a hint only if you are stuck. The model solution is for self-checking; your written work is not automatically marked.
Outline how to follow starch disappearance in an amylase reaction.
Give me a hint
Take timed samples and test each for remaining starch.
Compare my reasoning
- Mix amylase and starch and start timing consistently.
- At fixed intervals, test equal samples with iodine and record the colour.
Mix amylase and starch at controlled temperature and pH, starting the timer consistently. Remove equal samples at fixed intervals and add iodine. Record when the result changes from blue-black to orange-brown, indicating that starch is no longer detected.
Look for these in your work
- Timed sampling described
- Iodine colour interpretation correct
Starch is detected at 30 s but not at 45 s. Give the most precise justified time statement.
Give me a hint
Report the interval, not an unsupported exact time.
Compare my reasoning
- It remained detectable at 30 s.
- It was not detected by 45 s.
The starch fell below the iodine test’s detection level sometime after 30 s and by 45 s. These samples do not identify an exact disappearance time.
Look for these in your work
- Both observations used
- Uncertainty stated
A student uses pH 5 in one run and pH 8 in a comparison while claiming to test a different starch concentration. Explain the problem.
Give me a hint
Identify the uncontrolled variable and its effect.
Compare my reasoning
- pH also changed.
- pH can alter enzyme activity, so the effect cannot be attributed only to substrate concentration.
The pH is a confounding variable because it can affect amylase activity. Keep pH constant with a suitable buffer when testing the effect of starch concentration.
Look for these in your work
- Confound identified
- Effect on interpretation explained
Common mistakes
- Calling orange-brown iodine a positive starch result
- Claiming an exact disappearance time between samples
- Changing sampling volume or conditions across trials
What can you explain now?
Which reagent can indicate whether starch remains in a sample?
Compare with the explanation
Iodine solution
Starch gives a blue-black result with iodine.
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Make it stick
How does iodine reveal starch disappearance during amylase action, and what limits the timing estimate?
If you needed the solution, close it and solve the problem again from a blank page. A correct answer is stronger when you can explain the reason for each step.